The interleukin-7 (IL-7) signaling pathway plays essential roles in modeling the extracellular matrix and the development and homeostasis of B and T cells. Aberrant IL-7 signaling has been implicated in severe combined immunodeficiency, autoimmune conditions, and cancers. The IL-7 signal is triggered when soluble IL-7 interacts with two cell surface cytokine receptors, interleukin-7 receptor (IL-7R) and the common gamma- chain. In the previous funding period, we determined the structural, energetic, and cell biological features of the interaction of IL-7 with IL-7R. In this funding period, we will dissect the moleculr mechanisms of the interactions of IL-7 with glycosaminoglycans (e.g. heparin/heparin sulfate) and the common gamma-chain receptor with IL-7 and IL-7R. Preliminary studies of these interactions reveal the formation of unexpected non- activating complexes, either unique to IL-7 signaling or unidentified for the other common gamma-chain family members. Specifically, glycosaminoglycan binding induces dimer formation of IL-7, and common gamma- chain receptor can bind to IL-7 independent of IL-7R and to IL-7R independent of IL-7. Further studies will include three-dimensional x-ray crystallographic structures of the non-activating IL-7/GAG, IL-7/common gamma-chain, IL-7R/common gamma-chain complexes, as well as the activating IL-7/IL-7R/common gamma- chain ternary complex. The binding determinants of these epitopes also will be determined using site-directed scanning mutagenesis and various biophysical methods, including isothermal titration calorimetry and surface plasmon resonance. The structural and biophysical studies of non-activating and activating complexes ultimately will be used to develop models that explain normal and aberrant IL-7 signaling mechanisms and generate novel protein reagents and therapeutics that treat the numerous diseases associated with impaired IL-7 signaling.